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AMPK Is the Crucial Target for the CDK4/6 Inhibitors Mediated Therapeutic Responses in PANC-1 and MIA Paca-2 Pancreatic Cancer Cell Lines

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AMPK Is the Crucial Target for the CDK4/6 Inhibitors Mediated Therapeutic Responses in PANC-1 and MIA Paca-2 Pancreatic Cancer Cell Lines Article AMPK Is the Crucial Target for the CDK4/6 Inhibitors Mediated Therapeutic Responses in PANC-1 and MIA PaCa-2 Pancreatic Cancer Cell Lines Bortecine Sevgin 1,† , Merve Nur Coban 1,† , Özge Rencuzogullari 1, Ajda Coker-Gurkan 2 , Pinar Obakan-Yerlikaya 1 , Pinar Uysal Onganer 3 and Elif Damla Arisan 4,* 1 Department of Molecular Biology and Genetics, Atakoy Campus, Istanbul Kultur University, 34156 Istanbul, Turkey; [email protected] (B.S.); [email protected] (M.N.C.); [email protected] (Ö.R.); [email protected] (P.O.-Y.) 2 Department of Molecular Biology and Genetics, Biruni University, 34010 Istanbul, Turkey; [email protected] 3 Cancer Research Group, School of Life Sciences, College of Liberal Arts and Sciences, University of Westminster, 115 New Cavendish Street, London W1W 6UW, UK; [email protected] 4 Institute of Biotechnology, Gebze Technical University, 41400 Gebze, Turkey * Correspondence: [email protected] † These authors contributed equally to this work. Abstract: The survival rate of pancreatic ductal adenocarcinoma (PDAC) patients is short, and PDAC is a cancer type that ranks fourth in the statistics regarding death due to cancer. Mutation in the KRAS gene, which plays a role in pancreatic cancer development, activates the PI3K/AKT/mTOR Citation: Sevgin, B.; Coban, M.N.; signaling pathway. The activity of the AMPK as a cellular energy sensor is one of the fundamental Rencuzogullari, Ö.; mechanisms that can induce effective therapeutic responses against CDK4/6 inhibitors via adjusting Coker-Gurkan, A.; the cellular and tumor microenvironment stress management. The phosphorylation of AMPKα at Obakan-Yerlikaya, P.; the different phosphorylation residues such as Thr172 and Ser 377 causes metabolic differentiation Uysal Onganer, P.; Arisan, E.D. in the cells following CDK4/6 inhibitor treatment in accordance with an increased cell cycle arrest AMPK Is the Crucial Target for the and senescence under the control of different cellular players. In this study, we examined the CDK4/6 Inhibitors Mediated Therapeutic Responses in PANC-1 competencies of the CDK4/6 inhibitors LY2835219 and PD-0332991 on the mechanism of cell survival and MIA PaCa-2 Pancreatic Cancer and death based on AMPK signaling. Both CDK4/6 inhibitors LY2835219 and PD-0332991 modulated Cell Lines. Stresses 2021, 1, 48–68. different molecular players on the PI3K/AKT/mTOR and AMPK signaling axis in different ways to https://doi.org/10.3390/ reduce cell survival in a cell type dependent manner. These drugs are potential inducers of apoptosis stresses1010005 and senescence that can alter the therapeutic efficacy cells. Received: 19 January 2021 Keywords: pancreatic ductal adenocarcinoma; PD-0332991; LY2835219; PI3K/AKT/mTOR and Accepted: 23 February 2021 AMPK signaling axis; cell cycle Published: 18 March 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in 1. Introduction published maps and institutional affil- Pancreatic ductal adenocarcinoma (PDAC) is the most prevalent type of pancreatic iations. cancer that accounts for about 85% of pancreatic tumors. PDAC is the ninth most common cancer in women and the tenth most common cancer in men. It is also the fourth leading cause of cancer death [1]. In pancreatic cancer patients, 70% of whom are between the ages of 55 and84 and mostly men [2,3], the mutation in the oncogene KRAS Copyright: © 2021 by the authors. (proto-oncogene, GTPase) gene, which plays a role in the development of PDAC, acti- Licensee MDPI, Basel, Switzerland. vates the Cyclin-Dependent Kinases (CDK) and the mTOR signaling pathway associated This article is an open access article with increased cell survival [4,5]. Molecular profiling studies have shown that KRAS distributed under the terms and mutations and the CDK inhibitor 2A (CDKN2A or INK 4A/ARF), a tumor suppressor conditions of the Creative Commons gene, is inactivated in PDAC. The CDKN2A gene encodes the p16INK4a protein, a Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ potent inhibitor of CDK4 and CDK6 [6,7]. The loss of CDK2NA (p16) and tumor suppres- 4.0/). sor TP53 in the cell cycle causes uncontrolled cell proliferation. Moreover, accumulating Stresses 2021, 1, 48–68. https://doi.org/10.3390/stresses1010005 https://www.mdpi.com/journal/stresses Stresses 2021, 1 49 mutations in KRAS, TP53, MYC (myelocytomatosis oncogene cellular homologous), and LKB1 (liver kinase B1) play a role in glucose metabolism, which affects cell mainte- nance [5,8–10]. Therefore, altered metabolic machinery in PDAC cells is promising to find a new cure for successful disease management. LKB1 acts as a master upstream kinase directly phosphorylating AMP-activated protein kinase (AMPK), defined as a well-known regulator of cellular energy homeostasis. The LKB1-AMPK pathway serves as a metabolic checkpoint in the cell, arresting cell growth in low intracellular nutrient and ATP levels, which is crucial for cancer prognosis [11,12]. Furthermore, CDK4 is activated in the disrupted cell cycle to inhibit AMPK activity via direct phosphorylation of the AMPK-α2 subunit [13,14]. Therefore, changes in the activity of AMPK play an es- sential role in tumor aggressiveness [15,16]. The decreased AMPK activity has been also shown to increase pancreatic cancer invasion and metastasis through the Heat Shock Protein -1 (HSF1)-dependent manner [17]. The PI3K/AKT/mTOR pathway plays a criti- cal role in controlling cell growth, proliferation, migration, and metabolism [18–20]. PI3K signaling negatively regulates phosphatase and tensin homolog (PTEN), which is altered in most cancers, including pancreatic cancer [20,21]. PTEN also acts as a tumor suppressor gene by exhibiting phosphatase protein activity. AMPK inhibits the PI3K/AKT/mTOR signaling to reduce cell proliferation through the attenuation of Cyclin D, CDK4, and CDK6 levels [22–24]. The activation of upstream mitogenic pathways that increase CDK4/6 activity creates resistance to therapy in most tumor types [25,26]. Therefore, the continuous activation of the cell cycle and related signaling pathways has enabled the use of CDK inhibitors as an effective therapeutic agents in cancer treatment [27]. Palbociclib (PD 0332991) and abemaciclib (LY2835219), known as the CDK4/6 inhibitors, are the therapeutic agents used in the treatment of advanced metastatic hormone receptor-positive (HR+)/HER2-negative breast cancer [28–30]. The effects of these small molecule inhibitors are investigated in many types of malignancies as they are essential in the regulation of various signaling mechanisms, either alone or combined therapy regimes [28]. LY2835219, which has a similar structural property to PD-0332991, is remarkable for crossing the blood brain barrier. Trials are ongoing to evaluate LY2835219 in cancer patients with brain metastases [31]. LY2835219 binds competitively to CDK4 and CDK6 at the ATP binding site and has been found to ex- hibit 14 times more specific critical inhibition activity compared to PD 0332991 [32]. It was reported in hepatocellular carcinoma that palbociclib induced the AMPK activity independently of CDK4/6 [33]. Mostly, CDK4 is accepted as the new metabolic sensor that antagonizes AMPK to mediate a metabolic switch. For this purpose, the CDK4/6 inhibitor mediated downstream metabolic alteration related to AMPK is important for understanding the drug responsiveness in different cancer cells. AMPK activation mediated mTORC1 inhibition possess essential role in the regulation of replicative senescence in different cell lines [34]. It is also well described that PD-0332991 and LY2835219 have been shown to prevent the phosphorylation of the retinoblastoma (RB) protein, a well-known tumor suppressor, thereby invoking cancer cell cycle arrest at the G1 phase [27,30,35,36]. Almost all CDK4/6 inhibitors have been shown to induce apoptosis in several types of tumors and are associated with canonical effects such as cell cycle inhibition and proliferation [29,37–39]. LY-2535219 and PD-0332991 have re- cently revealed new non-canonical functions, inducing reversible senescence, metabolic rearrangement, and immunomodulation [39,40]. The activation of senescence may be a promising novel approach for cancer treatment [41,42]. This study evaluated the therapeutic potential of CDK4/6 inhibitors based on differential AMPK activation status in PANC-1 and MIA PaCa-2 cells with different genetic and biological features. Both cells had homozygous deletions of CDKN2A and were highly metastatic. The expression level of PI3K and AMPK was higher in MIA PaCa-2 cells than PANC-1 cells [43]. Therefore, the effect of both CDK inhibitors LY- 2535219 and PD-0332991 on the inhibition of cell proliferation was determined through Stresses 2021, 1 50 PI3K/AKT/mTOR/AMPK signaling axis and senescence mechanism in pancreatic cancer cells. 2. Results 2.1. CDK4/6 Inhibitors Reduced Cell Viability and Proliferation in a Dose-Dependent Manner To examine the effect of LY2835219 and PD-0332991 on relative cell viability and colony-forming ability in PANC-1 and MIA PaCa-2 PDAC cells, we used MTT and colony formation assays. PANC-1 and MIA PaCa-2 cells were treated with LY2835219 and PD-0332991 at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 7.5 and 10 µM concentrations for 24 h. We found that increasing concentrations of LY2835219 and PD-0332991 decreased cell viability in a dose-dependent manner in PANC-1 and MIA PaCa-2 cells (Figure1A,B). Low concentra- tion of LY2835219 such as 1 µM and 2 µM treatment caused a significant decrease in cell viability by 50% (50 ± 1.4%; n = 4; p < 0.001) PANC-1 cell lines. Moreover, MIA PaCa-2 cells were more sensitive, and 1 µM and 2 µM treatment of LY2835219 further decreased cell viability by 50% and 70%, respectively. Therefore, the IC50 values of LY2835219 were 2 µM and 1 µM in PANC-1 and MIA PaCa-2 cells, respectively. Similarly, 2 µM and 3 µM PD-0332991 treatment reduced cell viability by 50% (50 ± 1.3%; n = 4; p < 0.001) in PANC-1 and MIA PaCa-2 cells [29,44,45].
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